Hello,
I am examining the phosphorylation of the MAP kinase ERK by immunofluorescence. In order to do this, I understand that serum-deprivation of cells will eliminate background phosphorylation levels. For how long should I serum-deprive the cells? Shall I keep them in low serum for a full passage prior to seeding in my assay plate? Or shall I seed them in the assay plate in low serum? Or shall I seed them in the assay plate in normal serum and then change to low serum after cell landing? How many hours of serum-deprivation will result in low background phosphorylation? I imagine that 24 hours at 1% serum should do the trick. Alternatively, should I eliminate serum altogether? After how long in low-serum or serum-free conditions will cells signal abnormally or arrest?
Thank you!
You can culture the cells in serum-free medium for 3 days and keep cells that are not deprived of serum as negative control for examining the phosphorylation of the MAP kinase ERK by immunofluorescence. For more detail kindly see the published paper in FEBS Lett. 2008 Aug 6;582(18):2703-8.
doi: 10.1016/j.febslet.2008.06.051. Epub 2008 Jul 9.
Serum starvation induces H2AX phosphorylation to regulate apoptosis via p38 MAPK pathway.
I culture the cells in serum, seed them in normal growth medium, and 2 hours before the experiment I change the medium to serum free. However, to stabilize the cells I add 0,1% BSA to this „serum-free“ medium. In my experience, this brings ERK phosphorylation down to very low levels, while keeping the cells healthy. (Published in Jurek et al, 2015 J Neurosci)
Actually, serum starvation depends on the type of cells (cancer cells need a long time or change the 0.2% FBS media frequently like once in 6/12 hours ). I recommend a frequent change in media could be a better option to see good phosphorylation for farther analysis. seed low number of cells and let them grow in 0.2% FBS media with frequent change, until they grow 70-80% confluent (cell number depends on the dish you choose). this method could give you better results.
- It is generally recommended avoid adding FBS with drug as it binds the proteins, affecting the availability of enzymes/proteins evaluation.
- Starvation for 1-6 hours can synchronize the cell cycle, it depends on cell line, for istance primary cells do not tollerate starvation for more than 2-3 hours.
Depending the type of cells and media using for starvation. Primary cells are satrved for 3-5 hours and cancer cell lines are starved for 24 hours
I agreed with other replies that it really depends on cell types.
- Cancer cells (over-night starvation)
- primary cells (4-5 hours)
- non-cancerous cells (can be upto 24 hrs)
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